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WO2018008435A1 - Procédé d'analyse d'acides nucléiques - Google Patents

Procédé d'analyse d'acides nucléiques Download PDF

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Publication number
WO2018008435A1
WO2018008435A1 PCT/JP2017/023323 JP2017023323W WO2018008435A1 WO 2018008435 A1 WO2018008435 A1 WO 2018008435A1 JP 2017023323 W JP2017023323 W JP 2017023323W WO 2018008435 A1 WO2018008435 A1 WO 2018008435A1
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Prior art keywords
nucleic acid
target
probe
sample
control
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Japanese (ja)
Inventor
光一郎 辻丸
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Dnaform KK
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Dnaform KK
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a nucleic acid analysis method.
  • a nucleic acid in a sample is amplified with a specific primer and the presence or absence of the amplification is detected, or the obtained amplification product and a specific probe are hybridized.
  • a method for detecting soybean is employed.
  • amplification of the nucleic acid is essential from the viewpoint of accuracy and sensitivity.
  • the analysis result obtained is the result of the amplified nucleic acid, so the amount of nucleic acid originally contained in the sample is unknown.
  • the sample amount is small, a sufficient signal cannot be obtained even if a probe is used. For this reason, for example, the use of a fluorescent reagent that binds non-specifically to double-stranded DNA has been attempted, but this method detects non-specific DNA because it is non-specific binding. Stays on.
  • an object of the present invention is to provide a nucleic acid analysis method that enables specific analysis of nucleic acid regardless of the amount of the sample even if the sample is not subjected to amplification treatment.
  • the first nucleic acid analysis method of the present invention comprises: A mixing step of mixing the biological sample and the control probe; and A detection step of detecting hybridization between the nucleic acid in the sample and the control probe in the mixture;
  • the control probe is A fluorogenic probe in which a signal generator is bound to a nucleic acid molecule;
  • the nucleic acid molecule is a nucleic acid molecule that hybridizes to a predetermined control nucleic acid that is constantly present in the living body,
  • the signal generating substance is a substance that generates a luminescent signal by hybridization to a target, and disappears by emitting from the target.
  • the detection step includes The control nucleic acid in the sample is detected by detecting the hybridization.
  • the second nucleic acid analysis method of the present invention comprises: A mixing step of mixing a sample derived from a living body and a target probe; and A detection step of detecting hybridization between the nucleic acid in the sample and the target probe in the mixture;
  • the target probe is A fluorogenic probe in which a signal generator is bound to a nucleic acid molecule;
  • the nucleic acid molecule is a nucleic acid molecule that hybridizes to a target nucleic acid,
  • the signal generating substance is a substance that generates a luminescent signal by hybridization to a target, and disappears by emitting from the target.
  • the detection step includes The target nucleic acid in the sample is detected by detecting the hybridization.
  • the nucleic acid analysis method of the present invention for example, even for a sample that has not been subjected to amplification treatment, the nucleic acid can be specifically analyzed by using the fluorogenic probe regardless of the amount of the sample. .
  • the first nucleic acid analysis method of the present invention is as follows.
  • the control probe is A fluorogenic probe in which a signal generator is bound to a nucleic acid molecule;
  • the nucleic acid molecule is a nucleic acid molecule that hybridizes to a predetermined control nucleic acid that is constantly present in the living body,
  • the signal generating substance is a substance that generates a luminescent signal by hybridization to a target, and disappears by emitting from the target.
  • the detection step includes The control nucleic acid in the sample is detected by detecting the hybridization.
  • the control nucleic acid can be detected without performing the amplification treatment of the sample and without being influenced by the amount of the sample.
  • the sample is detected by detection of the control nucleic acid as described later. It is also possible to estimate the number of cells inside. In fields such as diagnosis, it is important to know the amount per cell. According to the present invention, since the number of cells in a sample can be estimated, it can be said that this is a very useful method in the field of diagnosis and the like.
  • the nucleic acid sample is a nucleic acid sample that has not been amplified.
  • control nucleic acid is DNA or RNA.
  • control nucleic acid is DNA or RNA encoding actin.
  • control probe has at least two fluorescent atomic groups exhibiting an exciton effect as the signal generating substance per molecule.
  • the first nucleic acid analysis method of the present invention uses, for example, a plurality of the control probes for the same control nucleic acid in the mixing step, Each of the plurality of control probes has a nucleic acid molecule that hybridizes to a different region with respect to the control nucleic acid.
  • the first nucleic acid analysis method of the present invention further includes, for example, a total nucleic acid detection step and a control nucleic acid ratio calculation step
  • the total nucleic acid detection step includes: Detecting the total nucleic acid in the sample
  • the ratio calculating step includes: Based on the detection result of the total nucleic acid and the detection result of the control nucleic acid, the ratio of the control nucleic acid in the total nucleic acid is calculated.
  • the first nucleic acid analysis method of the present invention further includes, for example, a calculation step of calculating the number of cells, the amount of tissue, or the amount of body fluid of the sample based on the correlation and the detection result of the control nucleic acid in the sample.
  • Including The correlation is a correlation between the number of cells, the amount of tissue or the amount of body fluid, and the amount of the control nucleic acid contained therein.
  • a mixing step of further mixing the sample and the target probe and A detection step of detecting hybridization between the nucleic acid in the sample and the target probe in the mixture;
  • the target probe is A probe that hybridizes to a target nucleic acid,
  • the detection step includes The target nucleic acid in the sample is detected by detecting the hybridization.
  • the target probe is: A fluorogenic probe in which a signal generator is bound to a nucleic acid molecule;
  • the nucleic acid molecule is a nucleic acid molecule that hybridizes to the target nucleic acid,
  • the signal generating substance is a substance that generates a luminescent signal by hybridization to a target, and disappears by emitting from the target.
  • the luminescent signal of the signal generating substance in the control probe and the luminescent signal of the signal generating substance in the target probe have different fluorescence characteristics.
  • the target probe has at least two fluorescent atomic groups exhibiting an exciton effect as the signal generating substance per molecule.
  • the first nucleic acid analysis method of the present invention uses, for example, a plurality of the target probes for the same target nucleic acid in the mixing step, Each of the plurality of target probes has a nucleic acid molecule that hybridizes to a different region with respect to the target nucleic acid.
  • the first nucleic acid analysis method of the present invention further includes, for example, a total nucleic acid detection step, and a target nucleic acid ratio calculation step
  • the total nucleic acid detection step includes: Detecting the total nucleic acid in the sample
  • the ratio calculating step includes: The ratio of the target nucleic acid in the total nucleic acid is calculated based on the detection result of the total nucleic acid and the detection result of the target nucleic acid.
  • the base having a pair of fluorescent atomic groups exhibiting the exciton effect is represented by the following formula (16), (16b), (17), or (17b). Has a structure.
  • the detection step is a detection step by melting curve analysis.
  • fluorogenic means, for example, that a signal is generated in a state of being specifically bound to a target and that the signal is lost in an unbound state, and the generation and disappearance of the signal is reversible.
  • analysis may be, for example, qualitative analysis or quantitative analysis.
  • the sample may be, for example, a sample that has not been amplified or a sample that has been amplified.
  • the present invention is particularly useful for analysis of a sample that has not been subjected to the former amplification treatment.
  • the sample is not particularly limited as long as it is derived from a living body.
  • the kind of the biological sample is not particularly limited, and examples thereof include body fluids, tissues, cells, and the like.
  • the body fluid include whole blood, plasma, blood such as serum, intraocular fluid such as aqueous humor, lymph, cerebrospinal fluid, tears, sweat, semen, saliva, mucus, urine, nasal discharge, and nasal swab Etc.
  • the tissue include intraocular tissues such as vitreous, and pathogenic tissues such as tumors.
  • the cells include pathogenic cells such as tumors, blood cells (for example, erythrocytes, leukocytes, etc.) and the like.
  • the kind of the living body is not particularly limited, and examples thereof include animals such as humans, non-human mammals (for example, cows, pigs, sheep, mice, rats, rabbits and horses), birds, and fish.
  • the control probe is a probe for control nucleic acid that is constantly present in a living body. Therefore, the control probe can be appropriately determined by setting the control nucleic acid according to the type of the sample to be analyzed, for example.
  • the control nucleic acid include a nucleic acid that is known to exist constitutively in the type of sample, and a nucleic acid that is known to exist constitutively in the future.
  • the control nucleic acid include nucleic acids encoding ⁇ -actin, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), ubiquitin and the like.
  • the amount per body fluid, the amount per tissue, or the amount per cell can be defined for the control nucleic acid such as actin depending on the type of animal (eg, human).
  • control nucleic acid to be analyzed may be, for example, DNA or RNA.
  • the control nucleic acid may be, for example, a single-stranded nucleic acid or a double-stranded nucleic acid.
  • any single-stranded nucleic acid may be set as a nucleic acid to which the control probe hybridizes.
  • the sense strand may be set as a hybridizing nucleic acid
  • the antisense strand may be set as a hybridizing nucleic acid.
  • the control probe is a fluorogenic probe in which a signal generating substance is bound to a nucleic acid molecule, and the nucleic acid molecule is a nucleic acid molecule that hybridizes to a predetermined control nucleic acid that is constantly present in the living body.
  • the nucleic acid molecule only needs to be able to hybridize to the control nucleic acid, and the sequence of the nucleic acid molecule can be appropriately designed according to the sequence of the control nucleic acid.
  • the nucleic acid molecule may be, for example, a nucleic acid molecule in which the control nucleic acid is single-stranded and forms a double strand by hybridization to the control nucleic acid, or the control nucleic acid is double-stranded, and the control nucleic acid It may be a nucleic acid molecule using Hoogsteen-type base pairing, which forms a triple strand by hybridization.
  • the nucleic acid molecule may be modified so that, for example, the 3 ′ end cannot be extended, and specifically, for example, the 3 ′ end may be chemically modified with a linker OH group.
  • the signal generating substance is a substance that generates a luminescent signal by hybridization to a target and disappears by dissociation from the target. Generation or disappearance of the signal may use, for example, FRET (Fluorescence resonance energy transfer) or may not use FRET.
  • FRET Fluorescence resonance energy transfer
  • Examples of the signal generating substance in the control probe include a fluorescent atomic group exhibiting an ethoxine effect.
  • the control probe preferably has, for example, at least two fluorescent atomic groups exhibiting an exciton effect as the signal generating substance per molecule (hereinafter also referred to as “E probe”).
  • the E probe can refer to, for example, Japanese Patent No. 4370385 and International Publication WO2014 / 013954 pamphlet.
  • the E probe is a probe into which two fluorescent atomic groups (for example, thiazole orange and the like) are introduced.
  • the E probe hardly emits fluorescence due to the exciton effect in which two fluorescent atomic groups form an exciplex, but hybridizes with the target to form a double-stranded state or 3
  • the two fluorescent atomic groups are separated from each other, and have the property of greatly emitting fluorescence by eliminating the exciton effect.
  • “E-probe” is a trade name of Danaform Co., Ltd. (“Eprobe” is a registered trademark), but “E-probe” in the present invention is given a product name of “E-probe” or “Eprobe”. The product may or may not be the same.
  • the binding position of the two fluorescent atomic groups is not particularly limited and can be set at any position.
  • the two fluorescent atoms may be bound to the same base in the nucleic acid molecule, or each may be bound to two adjacent bases.
  • the nucleic acid molecule is a nucleic acid molecule that forms a double strand with the control nucleic acid
  • at least one of the two fluorescent atoms is a base several bases inside from the 3 ′ end or the 5 ′ end of the nucleic acid molecule. It is preferable that the terminal base is the first, and it is preferable that the terminal base is bound to the base inside 3 bases.
  • the nucleic acid molecule is an oligonucleotide using Hoogsteen-type base pairs that form a triple strand with the control nucleic acid, for example, the two fluorescent atoms are at the 5 ′ end or the 3 ′ end, for example. It is preferred that they are bonded.
  • the two fluorescent atomic groups may be directly bonded to the oligonucleotide or indirectly bonded to the oligonucleotide, for example. In the latter case, the two fluorescent atomic groups are bonded to the oligonucleotide via, for example, a linker.
  • the fluorescent atomic group exhibiting the exciton effect is: (I) Two planar chemical structures in one molecule are not in the same plane but exist at a certain angle, but when the molecule intercalates or grooves binds to a nucleic acid, two planar chemical structures Fluorescence emission is caused by arranging the structures so that they are aligned in the same plane, (Ii) When the two or more dye molecules do not exhibit fluorescence due to the exciton effect caused by assembly in parallel, but when these molecules intercalate or groove bind to a target molecule, eg, a nucleic acid, It is composed of two or more dye molecule groups that generate fluorescence when the aggregated state is solved, or (Iii) When the two or more dye molecules do not exhibit fluorescence due to the exciton effect caused by the assembly in parallel, but when these molecules intercalate or groove bind to a target molecule, eg, a nucleic acid, It has the chemical structure of two or more dye molecules in which fluor
  • the base having a pair of fluorescent atomic groups exhibiting the exciton effect is represented by the following formula (16), (16b), (17), (17b), (18) or (18b), for example. It has a structure.
  • tautomers, stereoisomers, or salts thereof with respect to the structures represented by these formulas are also included in the structures in the present invention.
  • the structure represented by the following formulas having the atomic groups Z 11 and Z 12 exhibiting fluorescence may be referred to as “label structure”.
  • B is an atomic group having a natural nucleobase (adenine, guanine, cytosine, thymine or uracil) skeleton or an artificial nucleobase skeleton
  • E is (I) an atomic group having a structure derived from a deoxyribose skeleton, a ribose skeleton, or any of them, or (ii) an atomic group having a peptide structure or a peptoid structure
  • Z 11 and Z 12 are each an atomic group that exhibits fluorescence, and may be the same or different
  • L 1 , L 2 and L 3 are each a linker (a bridging atom or an atomic group)
  • the main chain length (the number of main chain atoms) is arbitrary
  • C, N, O, S, P and Si may or may not contain each, and in the main chain, single bond, double bond, triple bond, amide bond, ester
  • the main chain lengths (number of main chain atoms) of L 1 , L 2 and L 3 are each 2 or more It is preferable that it is an integer.
  • the upper limit of the main chain length (number of main chain atoms) of L 1 , L 2 and L 3 is not particularly limited, but is, for example, 100 or less, more preferably 30 or less, and particularly preferably 10 or less.
  • Z 11 and Z 12 are fluorescent atomic groups that exhibit an exciton effect.
  • the environmental change around the fluorescent dye when bound to the target sequence for example, the increase in fluorescence when a double helical structure is formed is large, and the target sequence can be detected more effectively.
  • Z 11 and Z 12 may be any fluorescent atomic group exhibiting an exciton effect, and are not particularly limited. More preferably, Z 11 and Z 12 are each independently a group derived from thiazole orange, oxazole yellow, cyanine, hemicyanine, other cyanine dyes, methyl red, azo dyes or derivatives thereof. In addition, groups derived from other known dyes can also be used as appropriate. Many fluorescent dyes that change fluorescence intensity by binding to nucleic acids such as DNA have been reported. In a typical example, ethidium bromide is known to exhibit strong fluorescence by intercalating into the double helix structure of DNA, and is frequently used for DNA detection.
  • fluorescent dyes capable of controlling the fluorescence intensity according to the microscopic polarity, such as pyrenecarboxamide and prodan.
  • the thiazole orange is a fluorescent dye in which a benzothiazole ring and a quinoline ring are connected by a methine group, and usually shows weak fluorescence, but emits strong fluorescence when intercalated into DNA having a double helix structure.
  • Other examples include dyes such as fluorescein and Cy3.
  • Z 11 and Z 12 are each independently an atomic group represented by any one of the following formulas (7) to (9).
  • X 1 and X 2 are S, Se or O; n ′′ is 0 or a positive integer; R 1 to R 10 and R 13 to R 21 are each independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,
  • R 11 and R 12 is a linking group that binds to L 1 or L 2 in the formulas (16), (17), (16b), (17b), (18), and (18b).
  • R 15 may be the same or different when a plurality of R 15 are present in the formula (7), (8) or (9), R 16 may be the same or different when there are a plurality of R 16 in the formula (7), (8) or (9), And X 1, X 2 and R 1 ⁇ R 21 in Z 11, and X 1, X 2 and R 1 ⁇ R 21 in Z 12, may be the same or different from each other.
  • the lower alkyl group is a linear or branched alkyl group having 1 to 6 carbon atoms, and the lower alkoxy group is 1 to carbon atoms. More preferably, it is a 6 straight-chain or branched alkoxy group.
  • the linking group is a polymethylene carbonyl group having 2 or more carbon atoms, and in the carbonyl group portion, the formulas (16), (16b), ( More preferably, it binds to L 1 or L 2 in 17), (17b), (18) and (18b).
  • the upper limit of the carbon number of the polymethylenecarbonyl group is not particularly limited, but is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, and particularly preferably 10 or less.
  • Z 11 and Z 12 are represented by the above formulas (7) to (9), for example, each independently is more preferably a group represented by the following formula (19) or (20).
  • X 1 represents —S— or —O—.
  • R 1 to R 10 , R 13 and R 14 each independently represent a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group or an amino group.
  • One of R 11 and R 12 represents a linking group bonded to L 1 and L 2 in the formulas (16), (17), (16b), (17b), (18) and (18b), and R The other of 11 and R 12 represents a hydrogen atom or a lower alkyl group.
  • Z 11 and Z 12 are each independently an atomic group represented by any one of the following chemical formulas.
  • n is a positive integer and is particularly preferably in the range of 2-6.
  • B may have a natural nucleobase skeleton. It may have a skeleton.
  • B is Py (pyrimidine ring), Py der. , Pu (purine ring), or Pu der. It is preferable that it is a structure represented by these.
  • the Py is an atomic group having a covalent bond bonded to E at the 1-position and a covalent bond bonded to the linker moiety at the 5-position among the 6-membered ring represented by the following formula (11). Yes, The Py der.
  • the Pu is an atomic group having a covalent bond bonded to E at the 9-position and a covalent bond bonded to the linker moiety at the 8-position among the condensed rings represented by the following formula (12). , The Pu der.
  • the oligonucleotide in the E probe is, for example, a nucleotide structure represented by the following chemical formula 106, 110, 113, 117, 120, 122, 123, 124 or 114-2, or a geometric isomer or stereoisomer thereof. It may contain at least one structure which is a body or a salt.
  • the linker length n is a positive integer and is preferably in the range of 2-6.
  • the number of the label structures included in the E probe is not particularly limited, but is, for example, about 1 to 100 and about 1 to 20. Further, in the E probe, the site including the label structure is not particularly limited.
  • the nucleic acid molecule in the control probe may be composed of any one of, for example, a natural nucleotide residue, a non-nucleotide residue, a modified nucleotide residue and a non-natural main skeleton, or any one kind or any two kinds Alternatively, the three types may be included, or the four types may be included.
  • the non-natural main skeleton is not particularly limited, and examples thereof include nucleic acids having LNA, PNA and modified phosphodiester bonds.
  • the modified nucleotide residue is not particularly limited and is a S-nucleotide residue, and the nucleotide residue may contain a sulfur atom (S) or may be modified with a sulfur atom (S).
  • the basic skeleton of the nucleic acid molecule in the control probe is not particularly limited.
  • the basic skeleton of the nucleic acid may be natural or artificially synthesized.
  • any nucleic acid may be used as long as it can form a base pair bond.
  • nucleic acid sample or a target nucleic acid sequence for example, it may function as a template for complementary strand synthesis.
  • the nucleic acid may be, for example, a nucleotide derivative partially or entirely composed of an artificial structure.
  • Examples of the artificial base constituting the nucleic acid include 2-amino-6- (N, N-dimethylamino) purinepyridin-2-one, 5-methylpyridin-2-one, 2-amino-6- (2-thienyl) purine, pyrrole-2-carbaldehyde, 9-Methylimidazo [(4,5) -b] pyridine, 5-iodo-2-oxo (1H) pyridine 2-oxo- (1H) pyridine, 2-amino-6- (2 -thiazolyl) purine, 7- (2-thienyl) -imidazo [4,5-b] pyridine and the like, but are not limited thereto.
  • nucleotide may be, for example, either deoxynucleotide or ribonucleotide
  • oligonucleotide and polynucleotide are, for example, either deoxynucleotide or ribonucleotide. It may be comprised from, and both may be included.
  • the number of bases constituting the nucleic acid is not particularly limited.
  • nucleic acid is generally synonymous with the term polynucleotide.
  • oligonucleotide is generally used as a term indicating a polynucleotide having a particularly small number of bases.
  • polynucleotide having a length of 2 to 100 bases, more generally about 2 to 50 bases is referred to as an “oligonucleotide”, but it is not limited to these numerical values.
  • polynucleotide is intended to include, in the first analytical method, for example, polynucleotides and oligonucleotides, as well as artificially synthesized nucleic acids such as peptide nucleic acids, morpholino nucleic acids, methyl phosphonate nucleic acids, S-oligonucleic acids.
  • the PNA peptide nucleic acid
  • the PNA generally has a structure in which the deoxyribose main chain of an oligonucleotide is replaced with a peptide main chain.
  • the peptide main chain include a repeating unit of N- (2-aminoethyl) glycine linked by an amide bond.
  • the base to be bound to the peptide main chain of PNA include thymine, cytosine, adenine, guanine, inosine, uracil, 5-methylcytosine, thiouracil and 2,6-diaminopurine, naturally occurring bases such as bromothymine, azaadenine And artificial bases such as azaguanine, but are not limited thereto.
  • the LNA is generally a nucleic acid having two circular structures in which the 2'-position oxygen atom and the 4'-position carbon atom of ribose are linked by a methylene bridge in the sugar-phosphate skeleton.
  • an oligonucleotide containing LNA anneals to DNA, the double-stranded conformation changes and thermal stability increases. Since LNA has a stronger binding force to nucleic acids than ordinary oligonucleotides, for example, more reliable and robust hybridization is possible depending on oligonucleotide design conditions.
  • the number of bases contained in the nucleic acid molecule in the control probe is not particularly limited, and is, for example, about 5 to 100, about 6 to 50, or about 6 to 25.
  • the mixing step is a step of mixing a biological sample and a control probe.
  • the amount of the biological sample is not particularly limited. According to the present invention, for example, in the case of a sufficient sample amount or a small sample amount, by using the control probe, it is possible to emit light in a sample without performing an amplification process.
  • the control nucleic acid can be detected.
  • the sample amount to be used may be, for example, in nanoliter order, microliter order, or milliliter order, for example, 0.1 nL to 1 mL, and specific examples include, for example, 0.1 nL to 10 nL, 10 nL to 100 nL. Yes, or 0.1 ⁇ L to 10 ⁇ L, 10 ⁇ L to 1000 ⁇ L.
  • the amount of the control probe added is not particularly limited, and can be appropriately set according to the sample amount, for example.
  • the addition amount is, for example, 1 ⁇ 10 ⁇ 12 to 1 ⁇ 10 ⁇ 9 ⁇ mol, 1 ⁇ 10 ⁇ 9 to 1 ⁇ 10 ⁇ 6 ⁇ mol, and 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 3 ⁇ mol with respect to 1 ⁇ L of the sample.
  • the control probe is preferably mixed with the sample, for example, as a probe reagent mixed in a solvent.
  • the concentration of the control probe is not particularly limited, for example, 1x10 -6 ⁇ 1x10 -3 ⁇ mol / L , 1x10 -3 ⁇ 1 ⁇ mol / L, 1 ⁇ 1x10 3 ⁇ mol / L.
  • the number of types of the control probe used in the mixing step is not particularly limited and can be appropriately set depending on the type or number of control nucleic acids to be set.
  • one type of control probe may be used, or two or more types of control probes may be used.
  • the mixing step a plurality of the control probes for the same control nucleic acid are used, and the plurality of control probes each have a nucleic acid molecule that hybridizes to a different region with respect to the control nucleic acid. It is preferable to have.
  • each of the plurality of control probes has a nucleic acid molecule that hybridizes to different regions, but preferably has the same signal generating substance and emits the same luminescent signal by hybridization.
  • a plurality of control probes can be hybridized to a plurality of locations of one type of control nucleic acid, and the total amount of luminescence signals by hybridization can be increased.
  • control nucleic acids when there are a plurality of control nucleic acids to be set, for example, one type of control probe may be used for each control nucleic acid, or two types of control probes may be used.
  • the control probe for each control nucleic acid preferably has a different signal luminescent substance, for example.
  • each control nucleic acid can be detected and separated by having different signal luminescent substances.
  • Different signal luminescent substances mean, for example, having different fluorescence properties, specifically, for example, having different excitation wavelengths, having different fluorescence wavelengths, or having different excitation wavelengths and different fluorescence wavelengths. Means that.
  • the mixed solution of the sample and the control probe is preferably processed at a temperature at which hybridization between the nucleic acid in the sample and the control probe is likely to occur (hybridization temperature), for example.
  • hybridization temperature a temperature at which hybridization between the nucleic acid in the sample and the control probe is likely to occur
  • the control nucleic acid is a double-stranded nucleic acid and is dissociated into a single-stranded nucleic acid and hybridized with the control probe
  • the sample is treated with a dissociation temperature, then mixed with the control probe,
  • the treatment may be performed at a hybridization temperature, or the mixture may be treated at a hybridization temperature after the mixture is treated at a dissociation temperature.
  • the hybridization temperature is, for example, 15 to 70 ° C.
  • the dissociation temperature is, for example, 90 to 100 ° C.
  • the detection step is a step of detecting hybridization between the nucleic acid in the sample and the control probe in the mixture.
  • the control nucleic acid in the sample can be detected by detecting the hybridization.
  • the method for detecting hybridization is not particularly limited, and can be appropriately determined depending on the signal generating substance in the control probe.
  • Examples of hybridization between the control probe and the control nucleic acid include detection by melting curve analysis.
  • the present invention may further include a total nucleic acid detection step and a control nucleic acid ratio calculation step for the sample.
  • the total nucleic acid detection step is a step of detecting total nucleic acid in the sample.
  • the method for detecting the total nucleic acid is not particularly limited, and examples thereof include a method for non-specifically detecting the nucleic acid in the sample.
  • an intercalator that intercalates into a double strand for example, SYBR (trademark) Green
  • SYBR trademark
  • the total nucleic acid detection step may be performed, for example, on the same system (the mixed solution) as the detection using the control probe, or may be performed on a different system, but the total amount of the sample to be used Can be reduced, the former is preferable. That is, it is preferable that the sample is mixed with the control probe and the intercalator, and both detection of hybridization with the control probe and detection of the total nucleic acid are performed.
  • the control probe and the intercalator may be added to the sample, for example, either first or simultaneously, and the detection of the hybridization and the detection of the total nucleic acid may be performed, for example, You may go first.
  • the ratio of the control nucleic acid in the total nucleic acid can be calculated based on the detection result of the total nucleic acid and the detection result of the control nucleic acid.
  • the present invention may further include a calculation step of calculating the number of cells, the amount of tissue, or the amount of body fluid of the sample based on the correlation and the detection result of the control nucleic acid in the sample.
  • the correlation is, for example, the relationship between the number of cells, the amount of tissue or the amount of body fluid, and the amount of the control nucleic acid contained therein.
  • the correlation can be obtained in advance from the amount of the control nucleic acid per target cell, tissue, or body fluid. it can. Based on the detection result of the control and the correlation, the number of cells, the amount of tissue, or the amount of body fluid in the sample can be calculated.
  • the target nucleic acid in the sample for example, even if the amount of the target nucleic acid can be analyzed, in diagnosis or the like, it is important to determine how many cells or the like are derived from. According to the present invention, by detecting the control nucleic acid, it is possible to further determine the amount of cells, tissues, or body fluids contained in the sample.
  • the present invention may further include a step of detecting a target nucleic acid for the sample.
  • the present invention further includes, for example, a mixing step of mixing the sample and the target probe, and a detection step of detecting hybridization between the nucleic acid in the sample and the target probe in the mixture.
  • the detection step the target nucleic acid in the sample can be detected by detecting the hybridization.
  • the present invention since the control nucleic acid that is constantly present in the living body can be detected, by further detecting the target nucleic acid, for example, the amount of the target nucleic acid relative to the control nucleic acid. Can be requested.
  • the target probe is not particularly limited, and a probe that hybridizes to a target nucleic acid can be used. However, like the control probe, a fluorogenic probe in which a signal generating substance is bound to a nucleic acid molecule is preferable.
  • the target probe is a fluorogenic probe
  • the nucleic acid molecule is a nucleic acid molecule that hybridizes to the target nucleic acid, and the signal generating substance generates a luminescent signal by hybridization to a target, It is a substance that loses its luminescence signal upon dissociation from the target, and the control probe and the target probe have different fluorescence characteristics.
  • control probe The description of the control probe can be used except that the target probe hybridizes to the target nucleic acid.
  • the number of types of the target probe is not particularly limited and can be appropriately set depending on the type or number of target nucleic acids to be set.
  • one type of target probe may be used, or two or more types of target probes may be used.
  • the mixing step a plurality of the target probes for the same target nucleic acid are used, and the plurality of target probes each have a nucleic acid molecule that hybridizes to a different region with respect to the target nucleic acid. It is preferable to have.
  • each of the plurality of target probes has a nucleic acid molecule that hybridizes to different regions, but preferably has the same signal generating substance and emits the same luminescent signal by hybridization.
  • a plurality of the target probes can be hybridized to a plurality of locations of one type of target nucleic acid, and the total amount of luminescence signals by the hybridization can be increased.
  • each target nucleic acid when a plurality of target nucleic acids are set, for example, one type of target probe may be used for each target nucleic acid, or two types of target probes may be used. It is preferable that the target probe for each target nucleic acid has a different signal luminescent substance, for example. Thus, each target nucleic acid can be detected and separated by having different signal luminescent substances.
  • Different signal luminescent substances mean, for example, having different fluorescence properties, specifically, for example, having different excitation wavelengths, having different fluorescence wavelengths, or having different excitation wavelengths and different fluorescence wavelengths. Means that.
  • the mixed solution of the sample and the target probe is preferably processed at a temperature at which hybridization between the nucleic acid in the sample and the target probe is likely to occur (hybridization temperature), for example.
  • hybridization temperature a temperature at which hybridization between the nucleic acid in the sample and the target probe is likely to occur
  • the sample is treated with a dissociation temperature, then mixed with the target probe,
  • the treatment may be performed at a hybridization temperature, or the mixture may be treated at a hybridization temperature after the mixture is treated at a dissociation temperature.
  • the hybridization temperature and the dissociation temperature are as described above, for example.
  • the method for detecting the hybridization is not particularly limited, and can be appropriately determined depending on, for example, the type of the target probe. As a specific example, it can be appropriately determined depending on the signal generating substance.
  • the target nucleic acid detection step may be performed, for example, on the same system (the mixed solution) as the detection using the control probe, or may be performed in another system, but the total amount of the sample to be used Can be reduced, the former is preferable. That is, it is preferable to mix the control probe and the target probe with a sample and perform both detection of hybridization with the control probe and detection of hybridization with the target probe.
  • the control probe and the target probe may be added to the sample, for example, either first or simultaneously, and detection of hybridization with the control probe and high detection with the target probe. Any detection of hybridization may be performed first, for example.
  • the present invention may further include a total nucleic acid detection step and a target nucleic acid ratio calculation step for the sample.
  • the total nucleic acid detection step is the same as described above, for example.
  • the ratio of the target nucleic acid in the total nucleic acid can be calculated based on the detection result of the total nucleic acid and the detection result of the target nucleic acid.
  • the ratio of the total nucleic acid, the control nucleic acid, and the target nucleic acid can be obtained for the sample.
  • control probe and optionally the target probe, and optionally the intercalator may be added to the reaction vessel, for example, or may be added in advance to the reaction vessel, or a necessary amount of the sample. May be held in a pipette tip to collect the. In the case of holding the pipette tip, for example, it is preferable to hold the sample so as to be released from the pipette tip by aspiration of the sample.
  • the first nucleic acid analysis method of the present invention will be described with reference to an example of detecting the control nucleic acid, the target nucleic acid and the total nucleic acid.
  • a control probe for the control nucleic acid, a target probe for the target nucleic acid, and an intercalator for the total nucleic acid are placed as dry reagents on the inner wall of the pipette tip for sample collection.
  • the control probe and the target probe are, for example, E probes each having different fluorescence characteristics.
  • the mixed liquid is discharged into a container, and the mixed liquid is hybridized with the control nucleic acid and the control probe, the target nucleic acid is hybridized with the target probe, and the intercalator is intercalated with respect to the total nucleic acid.
  • the control probe and the target probe have the same fluorescence wavelength at different excitation wavelengths, the control probe and the target probe are excited at the respective excitation wavelengths, and the fluorescence signals obtained by the respective excitation wavelengths are detected at the same fluorescence wavelength.
  • the control probe and the target probe have the same excitation wavelength and different fluorescence wavelengths, the control probe and the target probe are excited with the same excitation wavelength, and the fluorescence signal of each fluorescence wavelength is detected.
  • the amount of the control nucleic acid, the target nucleic acid, and the total nucleic acid is detected for the sample by the detection. Based on these results, the number of cells, the amount of tissue or the amount of body fluid in the sample can be calculated, and the ratio of the control nucleic acid to the total nucleic acid and the ratio of the target nucleic acid to the total nucleic acid or the control nucleic acid can be known. Can do.
  • the second nucleic acid analysis method of the present invention is as follows.
  • the target probe is A fluorogenic probe in which a signal generator is bound to a nucleic acid molecule;
  • the nucleic acid molecule is a nucleic acid molecule that hybridizes to a target nucleic acid,
  • the signal generating substance is a substance that generates a luminescent signal by hybridization to a target, and disappears by emitting from the target.
  • the detection step includes The target nucleic acid in the sample is detected by detecting the hybridization.
  • the target nucleic acid can be detected without performing the amplification process of the sample and without being influenced by the amount of the sample.
  • the description of the first nucleic acid analysis method can be used in the second nucleic acid analysis method of the present invention unless otherwise specified.
  • the sample is a sample that has not been amplified.
  • the target nucleic acid is DNA or RNA.
  • the target probe has at least two fluorescent atomic groups exhibiting an exciton effect as the signal generating substance per molecule.
  • the second nucleic acid analysis method of the present invention uses, for example, a plurality of the target probes for the same target nucleic acid in the mixing step, Each of the plurality of target probes has a nucleic acid molecule that hybridizes to a different region with respect to the target nucleic acid.
  • the second nucleic acid analysis method of the present invention further includes, for example, a total nucleic acid detection step and a target nucleic acid ratio calculation step
  • the total nucleic acid detection step includes: Detecting the total nucleic acid in the sample
  • the ratio calculating step includes: The ratio of the target nucleic acid in the total nucleic acid is calculated based on the detection result of the total nucleic acid and the detection result of the target nucleic acid.
  • the detection step is a detection step by melting curve analysis.
  • a fluorogenic probe is used as an actin probe, and actin DNA, which is a control nucleic acid, is detected from a non-amplified sample.
  • Probe for actin A DNA nucleic acid molecule having the following sequence is synthesized, and a signal generating substance is bound to 7 bases from the 3 ′ end so as to have the structure represented by the formula (113) to prepare an E probe. .
  • a probe reagent is prepared by mixing with Tris-NaCl buffer so that the concentration of E probe is 1 ⁇ 10 ⁇ 7 mol / L.
  • Probe sequence SEQ ID NO: 1): GGCGAACZGGTGGC (Z: labeled T base)
  • the nucleic acid analysis method of the present invention for example, even for a sample that has not been subjected to amplification treatment, the nucleic acid can be specifically analyzed by using the fluorogenic probe regardless of the amount of the sample. .

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Abstract

L'invention concerne un procédé d'analyse d'acides nucléiques qui permet une analyse, spécifique aux acides nucléiques, indépendamment d'une quantité d'acides nucléiques même pour un échantillon qui n'a pas subi un traitement d'amplification. Ce procédé d'analyse d'acides nucléiques comprend : une étape de mélange consistant à mélanger un échantillon dérivé d'un corps vivant et une sonde témoin ; et une étape de détection consistant à détecter, à partir du mélange, l'hybridation d'un acide nucléique dans l'échantillon et la sonde témoin. Le procédé d'analyse d'acides nucléiques est caractérisé en ce que : la sonde témoin est une sonde fluorogène dans laquelle des substances génératrices de signal sont liées à des molécules d'acide nucléique ; les molécules d'acide nucléique sont des molécules d'acide nucléique hybridables avec des acides nucléiques témoins prédéfinis constamment présents dans un corps vivant ; les substances génératrices de signal sont des substances qui génèrent un signal d'émission de lumière suite à l'hybridation à une cible et perdent le signal d'émission de lumière suite à la dissociation de la cible ; et dans l'étape de détection, les acides nucléiques témoins sont détectés à partir de l'échantillon par détection de l'hybridation.
PCT/JP2017/023323 2016-07-04 2017-06-26 Procédé d'analyse d'acides nucléiques Ceased WO2018008435A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008067694A (ja) * 2006-08-14 2008-03-27 Sony Corp 物質の検出等に有用な核酸鎖とその方法
JP2009526242A (ja) * 2006-02-13 2009-07-16 オルナトスキー オルガ 元素分析によって実施される遺伝子発現アッセイ
WO2013129457A1 (fr) * 2012-02-27 2013-09-06 東レ株式会社 Procédé de détection d'acides nucléiques
WO2014013954A1 (fr) * 2012-07-16 2014-01-23 株式会社ダナフォーム Sonde d'acide nucléique, procédé de conception d'une sonde d'acide nucléique, et procédé de détection de séquence cible
WO2014034818A1 (fr) * 2012-08-30 2014-03-06 株式会社ダナフォーム Procédé d'analyse d'acide nucléique cible, trousse et analyseur

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009526242A (ja) * 2006-02-13 2009-07-16 オルナトスキー オルガ 元素分析によって実施される遺伝子発現アッセイ
JP2008067694A (ja) * 2006-08-14 2008-03-27 Sony Corp 物質の検出等に有用な核酸鎖とその方法
WO2013129457A1 (fr) * 2012-02-27 2013-09-06 東レ株式会社 Procédé de détection d'acides nucléiques
WO2014013954A1 (fr) * 2012-07-16 2014-01-23 株式会社ダナフォーム Sonde d'acide nucléique, procédé de conception d'une sonde d'acide nucléique, et procédé de détection de séquence cible
WO2014034818A1 (fr) * 2012-08-30 2014-03-06 株式会社ダナフォーム Procédé d'analyse d'acide nucléique cible, trousse et analyseur

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